Evaluation of Metagenomic and Targeted Next-Generation Sequencing Workflows for Detection of Respiratory Pathogens from Bronchoalveolar Lavage Fluid Specimens

Abstract

Next-generation sequencing (NGS) workflows applied to bronchoalveolar lavage (BAL) fluid specimens could enhance the detection of respiratory pathogens, although optimal approaches are not defined. This study evaluated the performance of the Respiratory Pathogen ID/AMR (RPIP) kit (Illumina, Inc.) with automated Explify bioinformatic analysis (IDbyDNA, Inc.), a targeted NGS workflow enriching specific pathogen sequences and antimicrobial resistance (AMR) markers, and a complementary untargeted metagenomic workflow with in-house bioinformatic analysis.

Comparison of SARS-CoV-2 spike RNA sequences in feces and nasopharynx indicates intestinal replication

“…On average, Ct values for SARS-CoV-2 in the fecal samples with a complete spike genomic region sequence were 29.8 ± 2.1 and the Ct values of SARS-CoV-2 in the 14 corresponding NPH samples were 24 ± 5. Median [IQR] days between collection of NPH- and fecal samples was 1 [ 4 ]. The Alpha variant of SARS-CoV-2 was identified in the samples from 14 of the patients, whereas patient 9 was infected with the Beta variant.…”

“…The complete spike genomic region, known to more frequently mutate than other regions of the genome [5], was obtained for the strains in 15 of these fecal samples, and the NPH sample of corresponding patients, although one NPH sample was not recovered (patient 3). For another 9 fecal samples partial spike region [4]. The Alpha variant of SARS-CoV-2 was identified in the samples from 14 of the patients, whereas patient 9 was infected with the Beta variant.…”

“…A meta-analysis by Wong et al [1] found that a significant proportion of infected patients had positive SARS-CoV-2 RNA detected in their fecal specimens. Most studies on SARS-CoV-2 genetic divergences have focused on sequencing strains from NPH samples [2][3][4][5][6][7] and few studies have sequenced the SARS-CoV-2 genomes in stool samples from patients with COVID-19 [8][9][10][11][12]. A question remains if the viral RNA detected in stool samples is from replicating viruses in the intestines or from viruses having replicated in the respiratory tract.…”

Forty Years of Molecular Diagnostics for Infectious Diseases

“…Similar questions apply to metagenomic and other emerging NGS assays, with a need to systematically define their role alongside nonmolecular and targeted molecular assays ( 142 ). A prominent viewpoint, also in light of emerging data, is that these workflows might complement traditional methods, but certainly not yet replace them ( 143 ).…”

Detection of respiratory viruses in clinical samples

“…Recent review in clinical detection using bioinformatics methods only discusses about the application, method of sequencing techniques but they failed to discuss about the application of these techniques in novel viral pathogen detection. The main objective of the review are (i) to explain the role of NGS in identifying mutated DNA patterns of virus infected patients (ii) describe the contribution of these sequencing techniques in the detecting viral infections using several case studies (iii) bioinformatics challenges in using these techniques for clinical detection 1) [9].…”